1. Field of the Invention
The present invention relates to the field of biosensors and is more specifically concerned with methods for functionalizing sensing surfaces to be used in systems for measuring simultaneously several properties of one biomolecule, as well as measuring simultaneously the concentrations of a plurality of biomolecules in a sample. The invention furthermore also relates to sensor units containing such surfaces, a reagent kit for functionalizing the surfaces, and to the use thereof in the context of surface characterization of biomolecules.
2. Description of Related Art
According to Aizawa (1983) a biosensor is defined as being a unique combination of a receptor for molecular recognition, for example a selective layer with immobilized antibodies, and a transducer for transmitting the values measured. One group of such biosensors will detect the change which is caused in the optical properties of a surface layer due to the interaction of the receptor with the surrounding medium. Among such techniques may be mentioned especially ellipso-metry and surface plasmon resonance. In order for these types of techniques to work satisfactorily in actual practice, certain requirements have to be fulfilled--i.e., the requirement that the sensing surface employed can easily be derivatized so that it will then contain the desired receptor; and moreover that said surface will not produce any nonspecific binding, i.e., binding of components other than those that are intended, or that such binding in cases where it does occur will not furnish any significant contribution to the measuring signal. Some prior art in this field is exemplified below.
EP-A2-276 968 discloses a biograting for use in a light immunoassay comprising a polysilicon or single crystalline silicon surface having a biological diffraction grating design of an active binding reagent, such as an antibody, an antigen, a hapten, protein A, a lectin, biotin and avidin.
EP-A1-276 142 discloses a method of improving the sensitivity of assays based upon surface plasmon resonance by increasing the optical thickness of the optical surface, utilizing an additional complex forming reagent.
EP-A2-112 721 discloses an assay technique based upon observing the changes in the optical properties of a pre-formed surface, such as a grating, capable of binding the species to be assayed, the sample being applied to the surface by smearing. The pre-formed surface, such as a profiled plastic strip, may have a plurality of zones, each of which is coated with a different receptive material.
EP-Al-184 600 discloses a method for determining species dissolved in a liquid analyte by the use of an optical waveguide carrying a light signal and having one or more coatings of reactants specific to the species to be analyzed.
In somewhat simplified terms, the technique of surface plasmon resonance--by abbreviation SPR, as derived from the initials surface plasmon resonance--may be said to be a technique in which changes in the refractive index in a layer close to a thin free-electron metal film are detected by consequential changes in the intensity of a reflected p-polarized light beam (see for example Raether, H (1977)).
Thus, in this case, the sensing surface is a metal film with receptors or ligands as they will be called henceforth, these being generally molecules or molecular structures which interact selectively/specifically with one or more biomolecules.
The metal film is applied on a medium of a type that is suitable for the measuring method employed. In the case of SPR, this means that a transparent dielectric material, e.g., in the form of a glass plate, is used for directing a beam of light to the metal surface.
As regards the use of SPR procedures, most of the publications that have come forth up to now describe laboratory equipment for singular measurements. Although such a laboratory arrangement is apt to convey a general idea as to the potential of the method, all this is still a far cry from a commercially satisfactory instrument.
Most of the work done has focused on various methods for binding a particular biomolecule to the metal surface. In the first publication indicating the possibilities of SPR technology in biochemical analyses, Liedberg, B. et al (1983) have first adsorbed a monolayer of IgG onto a silver surface, and then adsorbed to said monolayer a layer of anti-IgG, in order to then study the effect in respect to the resultant change in the resonance angle. Others too, e.g., Cullen, D. C. et al. (1987/88), have utilized adsorption of biomolecules directly to a metal surface when studying immune complex formation in the IgG/anti-IgG system using the SPR technique with a gold-coated diffraction grating. EP 257955 describes a method according to which the metal film is coated with silica and optionally treated with a silanizing reagent; and in EP 202021, the metal film has been coated with an organic layer that may contain, for example, antibodies directed against a specific antigen and optionally bound by covalent bonds. According to EP 254575, an optical structure of the type such as is suitable for, e.g., SPR applications, may be produced by coating the metal film with a layer of an organic polymer, by means of the so-called "solvent casting technique". In a preferred embodiment, cellulose nitrate is employed, and a number of well-known methods are mentioned for binding bioselective ligands to the layer.
In other respects, the actual instrumentation equipment is described in only very brief terms, comprising an arrangement with a light source from which plane-polarized light in its plane of incidence is reflected from the metal surface and then intercepted by a detector, the output signal of which is usually recorded as a function of the angle of incidence. In this case, the metal film has been applied directly to a prism (see, e.g. Daniels, P. B. et al (1988)) or on a glass substrate, as a rule in the form of a microscope slide which is arranged in optical contact with the prism via immersion oil having a suitable refractive index (see, for example, GB 2197068). A variant form has been described by Webb et al. in EP 257955 according to which the glass substrate has been provided with a series of triangular projections from which rays of light from a lens system are coupled to the metal surface and then, after reflection, out to the detector system. In some cases it has been indicated that sample solutions are contacted with the sensing surface in a flow cell; but no actual practical directions are given as to how this is done.